8. Characterize “post 0.22 μm” and all the intermediates by (see
Note 12):
(a) Phenol sulfuric acid assay [15] for quantifying total sugar
content and HPAEC-PAD analysis for sugar composition,
content and yields of purification steps.
(b) Calculate average molecular weight distribution based on
the molar ratio of Rha to GlcNAc, considering that Rha is
present per each OAg chain repeating unit and that
GlcNAc is a unique sugar of the core region (see Note 13).
(c) HPLC-SEC for molecular size distribution.
(d) micro-BCA for protein content. Express it as ratio percentage relative to sugar content (w/w).
(e) Nucleic acids analysis by UV spectroscopy at wavelength
260 nm, assuming that a nucleic acid concentration of
50 μg/mL gives an OD260 of 1. Express it as a ratio
percentage relative to sugar content (w/w).
9. Characterize “post 0.22 μm” also by:
(a)
1 H NMR for OAg identity and O-acetyl content.
(b) Semicarbazide assay for KDO quantification (see Note
14).
(c) 2,4,6-trinitrobenzene sulfonate (TNBS) colorimetric
method for NH2 groups concentration [16].
l
Express it as a molar ratio % NH 2 groups to GlcNAc.
NH 2 groups can be present in OAg samples as pyrophosphoethanolamine residues in the core region [13].
(d) Chromogenic kinetic LAL (Limulus amoebocyte lysate)
to measure endotoxin level.
3.2 OAg Conjugation
to Carrier Protein
and Conjugate
Purification
Conjugation is performed by OAg activation at the terminus KDO
with ADH through its ketone group by reductive amination, followed by reaction of OAg-ADH with SIDEA and conjugation with
CRM 197 . Here the conjugation of S. paratyphi OAg is reported as
an example (Scheme 2). The same method can be applied to OAg
from other strains.
3.2.1 OAg Derivatization
1. Dry the desired amount of “post 0.22 μm” through rotating
evaporator (see Note 15).
2. Solubilize dried OAg (“post 0.22 μm”) in 100 mM AcONa
pH 4.5 at a concentration of 20–40 mg/mL. When OAg is
completely solubilized (see Note 16), add ADH and then
NaBH3CN as solids, both with a ratio 1.2:1 by weight with
respect to the OAg. Perform this operation within a
chemical hood.
3. Mix the solution at 30
C for 1 h.
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Francesca Micoli et al.
Note 12):
(a) Phenol sulfuric acid assay [15] for quantifying total sugar
content and HPAEC-PAD analysis for sugar composition,
content and yields of purification steps.
(b) Calculate average molecular weight distribution based on
the molar ratio of Rha to GlcNAc, considering that Rha is
present per each OAg chain repeating unit and that
GlcNAc is a unique sugar of the core region (see Note 13).
(c) HPLC-SEC for molecular size distribution.
(d) micro-BCA for protein content. Express it as ratio percentage relative to sugar content (w/w).
(e) Nucleic acids analysis by UV spectroscopy at wavelength
260 nm, assuming that a nucleic acid concentration of
50 μg/mL gives an OD260 of 1. Express it as a ratio
percentage relative to sugar content (w/w).
9. Characterize “post 0.22 μm” also by:
(a)
1 H NMR for OAg identity and O-acetyl content.
(b) Semicarbazide assay for KDO quantification (see Note
14).
(c) 2,4,6-trinitrobenzene sulfonate (TNBS) colorimetric
method for NH2 groups concentration [16].
l
Express it as a molar ratio % NH 2 groups to GlcNAc.
NH 2 groups can be present in OAg samples as pyrophosphoethanolamine residues in the core region [13].
(d) Chromogenic kinetic LAL (Limulus amoebocyte lysate)
to measure endotoxin level.
3.2 OAg Conjugation
to Carrier Protein
and Conjugate
Purification
Conjugation is performed by OAg activation at the terminus KDO
with ADH through its ketone group by reductive amination, followed by reaction of OAg-ADH with SIDEA and conjugation with
CRM 197 . Here the conjugation of S. paratyphi OAg is reported as
an example (Scheme 2). The same method can be applied to OAg
from other strains.
3.2.1 OAg Derivatization
1. Dry the desired amount of “post 0.22 μm” through rotating
evaporator (see Note 15).
2. Solubilize dried OAg (“post 0.22 μm”) in 100 mM AcONa
pH 4.5 at a concentration of 20–40 mg/mL. When OAg is
completely solubilized (see Note 16), add ADH and then
NaBH3CN as solids, both with a ratio 1.2:1 by weight with
respect to the OAg. Perform this operation within a
chemical hood.
3. Mix the solution at 30
C for 1 h.
278
Francesca Micoli et al.